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galaxy/tools/evolution/codingSnps.pl
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#!/usr/bin/perl -w
use strict;
#########################################################################
# codingSnps.pl
# This takes a bed file with the names being / separated nts
# and a gene bed file with cds start and stop.
# It then checks for changes in coding regions, reporting
# those that cause a frameshift or substitution in the amino acid.
# Output columns:
# chrom, start, end, allele as given (amb code translated)
# Gene ID from genes file, ref amino acid:variant amino acids,
# codon number, (in strand of gene)ref nt, refCodon:variantCodons
#########################################################################
my $seqFlag = "2bit"; #flag to set sequence type 2bit|nib
if (!@ARGV or scalar @ARGV < 3) {
print "Usage: codingSnps.pl snps.bed genes.bed (/dir/*$seqFlag|Galaxy build= loc=) [chr=# start=# end=# snp=# strand=#|-|+ keepColumns=1 synon=1 unique=1] > codingSnps.txt\n";
exit;
}
my $uniq = 0; #flag for whether want uniq positions
my $syn = 0; #flag for if want synonomous changes rather than non-syn
my $keep = 0; #keep old columns and append new ones
my $snpFile = shift @ARGV;
my $geneFile = shift @ARGV;
my $nibDir = shift @ARGV; #2bit or nib, depending on flag above
if ($nibDir eq 'Galaxy') { getGalaxyInfo(); }
my $col0 = 0; #bed like columns in default positions
my $col1 = 1;
my $col2 = 2;
my $col3 = 3;
my $strand = -1;
#column positions 1 based coming in (for Galaxy)
foreach (@ARGV) {
if (/chr=(\d+)/) { $col0 = $1 -1; }
elsif (/start=(\d+)/) { $col1 = $1 -1; }
elsif (/end=(\d+)/) { $col2 = $1 -1; }
elsif (/snp=(\d+)/) { $col3 = $1 -1; }
elsif (/keepColumns=1/) { $keep = 1; }
elsif (/synon=1/) { $syn = 1; }
elsif (/unique=1/) { $uniq = 1; }
elsif (/strand=(\d+)/) { $strand = $1 -1; } #0 based column
elsif (/strand=-/) { $strand = -99; } #special case of all minus
}
if ($col0 < 0 || $col1 < 0 || $col2 < 0 || $col3 < 0) {
print STDERR "ERROR column numbers are given with origin 1\n";
exit 1;
}
my @genes; #bed lines for genes, sorted by chrom and start
my %chrSt; #index in array where each chrom starts
my %codon; #hash of codon amino acid conversions
my $ends = 0; #ends vs sizes in bed 11 position, starts relative to chrom
my $ignoreN = 1; #skip N
my $origAll; #alleles from input file (before changes for strand)
my %amb = (
"R" => "A/G",
"Y" => "C/T",
"S" => "C/G",
"W" => "A/T",
"K" => "G/T",
"M" => "A/C",
"B" => "C/G/T",
"D" => "A/G/T",
"H" => "A/C/T",
"V" => "A/C/G",
"N" => "A/C/G/T"
);
fill_codon();
open(FH, "cat $geneFile | sort -k1,1 -k2,2n |")
or die "Couldn't open and sort $geneFile, $!\n";
my $i = 0;
while(<FH>) {
chomp;
if (/refGene.cdsEnd|ccdsGene.exonEnds/) { $ends = 1; next; }
push(@genes, "$_");
my @f = split(/\t/);
if (!exists $chrSt{$f[0]}) { $chrSt{$f[0]} = $i; }
$i++;
}
close FH or die "Couldn't close $geneFile, $!\n";
if ($ends) { print STDERR "WARNING using block ends rather than sizes\n"; }
#open snps sorted as well
my $s1 = $col0 + 1; #sort order is origin 1
my $s2 = $col1 + 1;
open(FH, "cat $snpFile | sort -k$s1,$s1 -k$s2,${s2}n |")
or die "Couldn't open and sort $snpFile, $!\n";
$i = 0;
my @g; #one genes fields, should be used repeatedly
my %done;
while(<FH>) {
chomp;
if (/^\s*#/) { next; } #comment
my @s = split(/\t/); #SNP fields
if (!@s or !$s[$col0]) { die "ERROR missing SNP data, $_\n"; }
my $size = $#s;
if ($col0 > $size || $col1 > $size || $col2 > $size || $col3 > $size) {
print STDERR "ERROR file has fewer columns than requested, requested columns (0 based) $col0 $col1 $col2 $col3, file has $size\n";
exit 1;
}
if ($strand >= 0 && $strand > $size) {
print STDERR "ERROR file has fewer columns than requested, requested strand in $strand (0 based), file has $size\n";
exit 1;
}
if ($s[$col1] =~ /\D/) {
print STDERR "ERROR the start point must be an integer not $s[$col1]\n";
exit 1;
}
if ($s[$col2] =~ /\D/) {
print STDERR "ERROR the start point must be an integer not $s[$col2]\n";
exit 1;
}
if ($s[$col3] eq 'N' && $ignoreN) { next; }
if (exists $amb{$s[$col3]}) { $s[$col3] = $amb{$s[$col3]}; }
if (($strand >= 0 && $s[$strand] eq '-') or $strand == -99) {
#reverse complement nts
$origAll = $s[$col3];
$s[$col3] = reverseCompAlleles($s[$col3]);
}else { undef $origAll }
if (!@g && exists $chrSt{$s[$col0]}) { #need to fetch first gene row
$i = $chrSt{$s[$col0]};
@g = split(/\t/, $genes[$i]);
if (scalar @g < 12) {
print STDERR "ERROR the gene file must be the whole genes in BED format\n";
exit 1;
}
}elsif (!@g) {
next; #no gene for this chrom
}elsif ($s[$col0] ne $g[0] && exists $chrSt{$s[$col0]}) { #new chrom
$i = $chrSt{$s[$col0]};
@g = split(/\t/, $genes[$i]);
}elsif ($s[$col0] ne $g[0]) {
next; #no gene for this chrom
}elsif ($s[$col1] < $g[1] && $i == $chrSt{$s[$col0]}) {
next; #before any genes
}elsif ($s[$col1] > $g[2] && ($i == $#genes or $genes[$i+1] !~ $s[$col0])) {
next; #after all genes on chr
}else {
while ($s[$col1] > $g[2] && $i < $#genes) {
$i++;
@g = split(/\t/, $genes[$i]);
if ($s[$col0] ne $g[0]) { last; } #end of gene
}
if ($s[$col0] ne $g[0] or $s[$col1] < $g[1] or $s[$col1] > $g[2]) {
next; #no overlap with genes
}
}
processSnp(\@s, \@g);
if ($uniq && exists $done{"$s[$col0] $s[$col1] $s[$col2]"}) { next; }
my $k = $i + 1; #check for more genes without losing data of first
if ($k <= $#genes) {
my @g2 = split(/\t/, $genes[$k]);
while (@g2 && $k <= $#genes) {
@g2 = split(/\t/, $genes[$k]);
if ($s[$col0] ne $g2[0]) {
undef @g2;
last; #not same chrom
}else {
while ($s[$col1] > $g2[2] && $k < $#genes) {
$k++;
@g2 = split(/\t/, $genes[$k]);
if ($s[$col0] ne $g2[0]) { last; } #end of chrom
}
if ($s[$col0] ne $g2[0] or $s[$col1] < $g2[1] or $s[$col1] > $g2[2]) {
undef @g2;
last; #no overlap with more genes
}
processSnp(\@s, \@g2);
if ($uniq && exists $done{"$s[$col0] $s[$col1] $s[$col2]"}) { last; }
}
$k++;
}
}
}
close FH or die "Couldn't close $snpFile, $!\n";
exit;
########################################################################
sub processSnp {
my $sref = shift;
my $gref = shift;
#overlaps gene, but maybe not coding seq
#inside cds
if ($sref->[$col1] + 1 < $gref->[6] or $sref->[$col2] > $gref->[7]) {
return; #outside of coding
}
#now check exon
my $i = 0;
my @st = split(/,/, $gref->[11]);
my @size = split(/,/, $gref->[10]);
if (scalar @st ne $gref->[9]) { return; } #cant do this gene #die "bad gene $gref->[3]\n"; }
my @pos;
my $in = 0;
for($i = 0; $i < $gref->[9]; $i++) {
my $sta = $gref->[1] + $st[$i] + 1; #1 based position
my $end = $sta + $size[$i] - 1; #
if ($ends) { $end = $size[$i]; $sta = $st[$i] + 1; } #ends instead of sizes
if ($end < $gref->[6]) { next; } #utr only
if ($sta > $gref->[7]) { next; } #utr only
#shorten to coding only
if ($sta < $gref->[6]) { $sta = $gref->[6] + 1; }
if ($end > $gref->[7]) { $end = $gref->[7]; }
if ($sref->[$col1] + 1 >= $sta && $sref->[$col2] <= $end) { $in = 1; }
elsif ($sref->[$col1] == $sref->[$col2] && $sref->[$col2] <= $end && $sref->[$col2] >= $sta) { $in = 1; }
push(@pos, ($sta .. $end)); #add exon worth of positions
}
#@pos has coding positions for whole gene (chr coors),
#and $in has whether we need to continue
if (!$in) { return; } #not in coding exon
if ((scalar @pos) % 3 != 0) { return; } #partial gene? not even codons
if ($sref->[$col3] =~ /^-+\/[ACTG]+$/ or $sref->[$col3] =~ /^[ACTG]+\/-+$/ or
$sref->[$col3] =~ /^-+$/) { #indel or del
my $copy = $sref->[$col3];
my $c = ($copy =~ tr/-//);
if ($c % 3 == 0) { return; } #not frameshift
#handle bed4 or any interval file
if (!$keep) {
print "$sref->[$col0]\t$sref->[$col1]\t$sref->[$col2]\t$sref->[$col3]";
print "\t$gref->[3]\tframeshift\n";
}else {
my @s = @{$sref};
print join("\t", @s), "\t$gref->[3]\tframeshift\n";
}
$done{"$sref->[$col0] $sref->[$col1] $sref->[$col2]"}++;
return;
}elsif ($sref->[$col1] == $sref->[$col2]) { #insertion
my $copy = $sref->[$col3];
my $c = ($copy =~ tr/\[ACTG]+//);
if ($c % 3 == 0) { return; } #not frameshift
#handle bed4 or any interval file
if (!$keep) {
print "$sref->[$col0]\t$sref->[$col1]\t$sref->[$col2]\t$sref->[$col3]";
print "\t$gref->[3]\tframeshift\n";
}else {
my @s = @{$sref};
print join("\t", @s), "\t$gref->[3]\tframeshift\n";
}
$done{"$sref->[$col0] $sref->[$col1] $sref->[$col2]"}++;
return;
}elsif ($sref->[$col3] =~ /-/) { #indel and sub?
return; #skip
}
#check for amino acid substitutions
my $s = $sref->[$col1] + 1;
my $e = $sref->[$col2];
my $len = $sref->[$col2] - $sref->[$col1];
if ($gref->[5] eq '-') {
@pos = reverse(@pos);
my $t = $s;
$s = $e;
$e = $t;
}
$i = 0;
my $found = 0;
foreach (@pos) {
if ($s == $_) {
$found = 1;
last;
}
$i++;
}
if ($found) {
my $fs = $i; #keep original start index
#have index where substitution starts
my $cp = $i % 3;
$i -= $cp; #i is now first position in codon
my $cdNum = int($i / 3) + 1;
my $ls = $i;
if (!defined $ls) { die "ERROR not defined ls for $fs $sref->[$col2]\n"; }
if (!@pos) { die "ERROR not defined array pos\n"; }
if (!defined $pos[$ls]) { die "ERROR not defined pos at $ls\n"; }
if (!defined $e) { die "ERROR not defined e for $pos[0] $pos[1] $pos[2]\n"; }
while ($ls <= $#pos && $pos[$ls] ne $e) {
$ls++;
}
my $i2 = $ls + (2 - ($ls % 3));
if ($i2 > $#pos) { return; } #not a full codon, partial gene?
if ($i2 - $i < 2) { die "not a full codon positions $i to $i2 for $sref->[3]\n"; }
my $oldnts = getnts($sref->[$col0], @pos[$i..$i2]);
if (!$oldnts) { die "Failed to get sequence for $sref->[$col0] $pos[$i] .. $pos[$i2]\n"; }
my @vars = split(/\//, $sref->[$col3]);
if ($gref->[5] eq '-') { #complement oldnts and revcomp vars
$oldnts = compl($oldnts);
if (!$oldnts) { return; } #skip this one
$oldnts = join('', (reverse(split(/ */, $oldnts))));
foreach (@vars) {
$_ = reverse(split(/ */)); #needed for indels
$_ = compl($_);
}
}
my $r = $fs - $i; #difference in old indexes gives new index
my @newnts;
my $changed = '';
foreach my $v (@vars) {
if (!$v or length($v) != 1) { return; } #only simple changes
my @new = split(/ */, $oldnts);
$changed = splice(@new, $r, $len, split(/ */, $v));
#should only change single nt
push(@newnts, join("", @new));
}
#now compute amino acids
my $oldaa = getaa($oldnts);
my $codon = "$oldnts:";
my @newaa;
my $change = 0; #flag for if there is a change
foreach my $v (@newnts) {
my $t = getaa($v);
if ($t ne $oldaa) { $change = 1; }
push(@newaa, "$t");
$codon .= "$v/";
}
$codon =~ s/\/$//;
if (!$change && $syn) {
if (!$keep) {
print "$sref->[$col0]\t$sref->[$col1]\t$sref->[$col2]\t$sref->[$col3]";
print "\t$gref->[3]\t$oldaa:", join("/", @newaa), "\t$cdNum\t$changed\t$codon\n";
}else {
my @s = @{$sref};
print join("\t", @s),
"\t$gref->[3]\t$oldaa:", join("/", @newaa), "\t$cdNum\t$changed\t$codon\n";
}
$done{"$sref->[$col0] $sref->[$col1] $sref->[$col2]"}++;
return;
}elsif ($syn) { return; } #only want synonymous changes
if (!$change) { return; } #no change in amino acids
if (!$keep) {
my $a = $sref->[$col3];
if (($strand >= 0 && $origAll) or $strand == -99) { $a = $origAll; }
print "$sref->[$col0]\t$sref->[$col1]\t$sref->[$col2]\t$a";
#my $minus = $changed; #in case minus strand and change back
#if ($gref->[5] eq '-') { $changed = compl($changed); } #use plus for ref
if (!$changed) { return; } #skip this one
print "\t$gref->[3]\t$oldaa:", join("/", @newaa), "\t$cdNum\t$changed\t$codon\n";
}else {
my @s = @{$sref};
if (($strand >= 0 && $origAll) or $strand == -99) { $s[$col3] = $origAll; }
print join("\t", @s);
#my $minus = $changed; #in case minus strand and change back
#if ($gref->[5] eq '-') { $changed = compl($changed); } #use plus for ref
if (!$changed) { return; } #skip this one
print "\t$gref->[3]\t$oldaa:", join("/", @newaa), "\t$cdNum\t$changed\t$codon\n";
}
$done{"$sref->[$col0] $sref->[$col1] $sref->[$col2]"}++;
}
}
sub getnts {
my $chr = shift;
my @pos = @_; #list of positions not necessarily in order
#list may be reversed or have gaps(introns), at least 3 bps
my $seq = '';
if (scalar @pos < 3) { die "too small region for $chr $pos[0]\n"; }
if ($pos[0] < $pos[1]) { #not reversed
my $s = $pos[0];
for(my $i = 1; $i <= $#pos; $i++) {
if ($pos[$i] == $pos[$i-1] + 1) { next; }
if ($seqFlag eq '2bit') {
$seq .= fetchSeq2bit($chr, $s, $pos[$i-1]);
}else {
$seq .= fetchSeqNib($chr, $s, $pos[$i-1]);
}
$s = $pos[$i];
}
if (length $seq != scalar @pos) { #still need to fetch seq
if ($seqFlag eq '2bit') {
$seq .= fetchSeq2bit($chr, $s, $pos[$#pos]);
}else {
$seq .= fetchSeqNib($chr, $s, $pos[$#pos]);
}
}
}else { #reversed
my $s = $pos[$#pos];
for(my $i = $#pos -1; $i >= 0; $i--) {
if ($pos[$i] == $pos[$i+1] + 1) { next; }
if ($seqFlag eq '2bit') {
$seq .= fetchSeq2bit($chr, $s, $pos[$i+1]);
}else {
$seq .= fetchSeqNib($chr, $s, $pos[$i+1]);
}
$s = $pos[$i];
}
if (length $seq != scalar @pos) { #still need to fetch seq
if ($seqFlag eq '2bit') {
$seq .= fetchSeq2bit($chr, $s, $pos[0]);
}else {
$seq .= fetchSeqNib($chr, $s, $pos[0]);
}
}
}
}
sub fetchSeq2bit {
my $chr = shift;
my $st = shift;
my $end = shift;
my $strand = '+';
$st--; #change to UCSC numbering
open (BIT, "twoBitToFa -seq=$chr -start=$st -end=$end $nibDir stdout |") or
die "Couldn't run twoBitToFa, $!\n";
my $seq = '';
while (<BIT>) {
chomp;
if (/^>/) { next; } #header
$seq .= uc($_);
}
close BIT or die "Couldn't finish twoBitToFa on $chr $st $end, $!\n";
return $seq;
}
sub fetchSeqNib {
my $chr = shift;
my $st = shift;
my $end = shift;
my $strand = '+';
$st--; #change to UCSC numbering
open (NIB, "nibFrag -upper $nibDir/${chr}.nib $st $end $strand stdout |") or die "Couldn't run nibFrag, $!\n";
my $seq = '';
while (<NIB>) {
chomp;
if (/^>/) { next; } #header
$seq .= $_;
}
close NIB or die "Couldn't finish nibFrag on $chr $st $end, $!\n";
return $seq;
}
sub compl {
my $nts = shift;
my $comp = '';
if (!$nts) { die "ERROR called compl with nts undefined"; }
foreach my $n (split(/ */, $nts)) {
if ($n eq 'A') { $comp .= 'T'; }
elsif ($n eq 'T') { $comp .= 'A'; }
elsif ($n eq 'C') { $comp .= 'G'; }
elsif ($n eq 'G') { $comp .= 'C'; }
elsif ($n eq 'N') { $comp .= 'N'; }
elsif ($n eq '-') { $comp .= '-'; } #deletion
else { $comp = undef; }
}
return $comp;
}
sub reverseCompAlleles {
my $all = shift;
my @nt = split(/\//, $all);
my $rv = '';
foreach my $n (@nt) {
$n = reverse(split(/ */, $n)); #needed for indels
$n = compl($n);
$rv .= "$n/";
}
$rv =~ s/\/$//;
return $rv;
}
sub getaa {
my $nts = shift; #in multiples of 3
my $aa = '';
my @n = split(/ */, $nts);
while (@n) {
my @t = splice(@n, 0, 3);
my $n = uc(join("", @t));
if (!exists $codon{$n}) { $aa .= 'N'; next; }
$aa .= $codon{$n};
}
return $aa;
}
sub fill_codon {
$codon{GCA} = 'Ala';
$codon{GCC} = 'Ala';
$codon{GCG} = 'Ala';
$codon{GCT} = 'Ala';
$codon{CGG} = 'Arg';
$codon{CGT} = 'Arg';
$codon{CGC} = 'Arg';
$codon{AGA} = 'Arg';
$codon{AGG} = 'Arg';
$codon{CGA} = 'Arg';
$codon{AAC} = 'Asn';
$codon{AAT} = 'Asn';
$codon{GAC} = 'Asp';
$codon{GAT} = 'Asp';
$codon{TGC} = 'Cys';
$codon{TGT} = 'Cys';
$codon{CAG} = 'Gln';
$codon{CAA} = 'Gln';
$codon{GAA} = 'Glu';
$codon{GAG} = 'Glu';
$codon{GGG} = 'Gly';
$codon{GGA} = 'Gly';
$codon{GGC} = 'Gly';
$codon{GGT} = 'Gly';
$codon{CAC} = 'His';
$codon{CAT} = 'His';
$codon{ATA} = 'Ile';
$codon{ATT} = 'Ile';
$codon{ATC} = 'Ile';
$codon{CTA} = 'Leu';
$codon{CTC} = 'Leu';
$codon{CTG} = 'Leu';
$codon{CTT} = 'Leu';
$codon{TTG} = 'Leu';
$codon{TTA} = 'Leu';
$codon{AAA} = 'Lys';
$codon{AAG} = 'Lys';
$codon{ATG} = 'Met';
$codon{TTC} = 'Phe';
$codon{TTT} = 'Phe';
$codon{CCT} = 'Pro';
$codon{CCA} = 'Pro';
$codon{CCC} = 'Pro';
$codon{CCG} = 'Pro';
$codon{TCA} = 'Ser';
$codon{AGC} = 'Ser';
$codon{AGT} = 'Ser';
$codon{TCC} = 'Ser';
$codon{TCT} = 'Ser';
$codon{TCG} = 'Ser';
$codon{TGA} = 'Stop';
$codon{TAG} = 'Stop';
$codon{TAA} = 'Stop';
$codon{ACT} = 'Thr';
$codon{ACA} = 'Thr';
$codon{ACC} = 'Thr';
$codon{ACG} = 'Thr';
$codon{TGG} = 'Trp';
$codon{TAT} = 'Tyr';
$codon{TAC} = 'Tyr';
$codon{GTC} = 'Val';
$codon{GTA} = 'Val';
$codon{GTG} = 'Val';
$codon{GTT} = 'Val';
}
sub getGalaxyInfo {
my $build;
my $locFile;
foreach (@ARGV) {
if (/build=(.*)/) { $build = $1; }
elsif (/loc=(.*)/) { $locFile = $1; }
}
if (!$build or !$locFile) {
print STDERR "ERROR missing build or locfile for Galaxy input\n";
exit 1;
}
# read $locFile to get $nibDir (ignoring commets)
open(LF, "< $locFile") || die "open($locFile): $!\n";
while(<LF>) {
s/#.*$//;
s/(?:^\s+|\s+$)//g;
next if (/^$/);
my @t = split(/\t/);
if ($t[0] eq $build) { $nibDir = $t[1]; }
}
close(LF);
if ($nibDir eq 'Galaxy') {
print STDERR "Failed to find sequence directory in locfile $locFile\n";
}
$nibDir .= "/$build.2bit"; #we want full path and filename
}
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